The preparation of oligonucleotide conjugates is generally accomplished through the use an oligonucleotide modified with a primary amine (Agrawal, S. (1994) Functionalization of oligonucleotides with amino groups and attachment of amino specific reporter groups. Methods in Molecular Biology 26; Protocols for Oligonucleotide Conjugates. (S. Agarwal, Ed.) pp. 73-92, Humana Press, Totowa, N.J. (Review), Meyers, R. (1994) Incorporation of Modified Bases into Oligonucleotides. Methods in Molecular Biology 26; Protocols for Oligonucleotide Conjugates. (S. Agarwal, Ed.) pp. 93-120, Humana Press, Totowa, N.J. (Review)). In most cases, amide or thiourea bonds are formed with conjugars containing an activated carboxyl or isothiocynate (ITC) functionality.
Although functionalization of many conjugars is routine, a number of conjugars have proved to be very difficult to transform into activated carboxyl or ITC derivatives either because of the complex synthesis involved or the inherent instability of the final compound. In an effort to circumvent these difficulties the coupling partners have been reversed placing the carboxylic acid function on the oligonucelotide, and the amine on the conjugar. The literature contains several examples of 5' terminal oligonucleotide linkers that contain a carboxyl funtionality. Unfortunately these methods have several disadvantages. Kremsky et al. ((1987) Immobilization of DNA via oligonucleotides containing and aldehyde or carboxylic acid group at the 5' terminus. Nucleic Acids Research 15, 2891-2909), describe conjugation with a protected 5' terminal oligonucleotide carboxyl group requiring cleavage of the methyl ester protecting group, followed by in situ activation with N-hydroxysuccinimide ("NHS") and a coupling reagent to achieve conjugation. The disadvantage of this method is the number of steps required to achieve the desired conjugate.
In another approach, the protecting group is a benzyl ester, which can be directly coupled to an amine (Endo, M., Gaga, Y., and Komiyama, M., (1994) A novel phosphoramidite for the site-selective introduction of functional groups into oligonucleotides via versatile tethers. Tetrahedron Letter 33, 3879-3882). However, this procedure calls for conditions that require treatment with a primary amine for 48 hours and is incompatible with base sensitive oligonucleotides, such as methylphosphonates (Hogrefe, R. I., Vaghefi, M. M., Reynolds, R. A., Young, K. M., and Arnold, L. J. Jr. (1993) Deprotection of methylphosphonate oligonucleotides using a novel one-pot procedure. Nucleic Acids Research 21, 2031-2038).
A third approach describes the formation of a phosphoramidate bond between a 3' or 5' phosphorylated oligonucleotide and an amino acid, followed by subsequent activation of the carboxyl moiety with carbodiimide (Gottikh, M., Asseline, U., and Thoung, N. T. (1990) Synthesis of oligonucleotides containing a carboxyl group at either their 5' end or their 3' end and their subsequent derivitization by an intercalating agent. Tetrahedron Letters 31, 6657-6660). The number of steps required to prepare these conjugates makes these methods generally undesirable.
Consequently there is a need for a fast and effective means of forming conjugating oligonucleotides to a variety of molecules while requiring a minimal amount of post-synthetic chemistry. In particular linker compositions are needed that may be synthesized from primary amine containing compounds that are relatively easy to obtain; comprise a non-labor intensive and efficient means for attachment to an oligonucleotide such as a phosphoramidite reagent that can be coupled directly to the 5' terminus; that are compatible with the conditions of oligonucleotide synthesis and that provide a highly reactive functionality, such as a succinimydyl or pentafluorophenyl ester. In addition, linker compositions that may be utilized in conjugations performed on support bound linker-modified oligonucleotides prior to deprotection and cleavage are particularly preferred.